Candidate and Environment: Dr. Khare's long-term goal is to study microbial interactions between biomedically relevant bacteria, to understand how such interactions affect human physiology and behavior. Her strong background in genetics and microbial research from her graduate studies, combined with her extensive training in bacterial systems biology during her current postdoctoral career provide her with a solid foundation to achieve this goal. The proposal outlined for the K99/R00 award will allow Dr. Khare to develop expertise in various novel systems, techniques and data analysis procedures critical for her future research, in addition to supporting her postdoctoral research on microbial interactions that will help to launch her career as an independent researcher. The training phase of the K99/R00 award is to be completed at the Joint Centers for Systems Biology in the Columbia University Medical Center campus, where Dr. Khare has access to various shared resources and facilities crucial for her research. The mentor's lab and department provide an opportunity for training in systems biology, as well as access to courses, seminars and collaborations with scientists from other research groups. Research: Interactions between bacterial species are a crucial, but understudied aspect of all microbial behaviors including survival and growth in niches within the human body, as well as multi-species infections. This research proposal aims to lay down a framework for the study of complex multispecies systems. It outlines an interdisciplinary approach to analyze health-relevant interactions between bacterial species and identify key molecular factors and mechanisms involved in these interactions. Pseudomonas aeruginosa is an opportunistic pathogen that causes numerous infections in immuno-compromised individuals, and is commonly found in co-infections with other bacteria including Staphylococcus aureus; however, the molecular details of its interactions with other bacterial species are not well-studied. These interactions likely play a crucial role in determining the outcome and dynamics of P. aeruginosa infections, and co-infection progression, and this research proposal aims to carry out an in-depth characterization of such interactions. Dr. Khare's preliminary data show that P. aeruginosa secretes molecules that have a growth-inhibitory effect on Escherichia coli, a gram-negative model organism, commensal and occasional human pathogen, as well as S. aureus, a pathogenic gram-positive bacterium. Biochemical and genomic analyses have identified several components of this growth inhibition: siderophore-mediated iron sequestration affecting both species, growth inhibition of E. coli by the phenazine virulence factors, and that of S. aureus by quinolones, and other unidentified factors. Further, Dr. Khare's data shows that P. aeruginosa can sense the presence of S. aureus, and respond by the secretion of additional antimicrobial factors. The goal of this proposal is to systematically delineate the molecular mechanisms underlying these interactions. The research planned for the K99 training phase is focused on characterizing the molecular mechanism of phenazine action in E. coli, and quinolone action in S. aureus. The molecular mechanism of action of the phenazine virulence factors will be elucidated by testing well-defined hypotheses formulated based on existing literature and Dr. Khare's preliminary data, using a variety of specific cellular assays. Further, experimental evolution techniques will be used to identify mutational trajectories that enable E. coli to resist this growth inhibition, giving insights into how bacteria survive as they are exposed to P. aeruginosa antimicrobials in various environments including human co-infections. The effect of quinolones on S. aureus will be studied similarly, using transcriptional analyses, screening of defined transposon insertion libraries and a collection of clinical isolates, as well as cellular assays based on specific hypotheses. This research should be completed early in the R00 phase. The last specific aim of this proposal, to be addressed during the independent phase, focusses on identifying the molecular pathways of P. aeruginosa sensing of and response to S. aureus, using a combination of biochemical and transcriptional analyses, as well as mutant libraries and specific reporter systems. This research will provide significant molecular insights into how P. aeruginosa senses and responds to foreign species, how its presence adversely affects other bacterial species in various environments, and the potential evolutionary trajectories available to these 'target' bacteria to overcome this antagonism, and is a first step towards understanding how stable microbial communities are formed and sustained. Further, the integrated experimental framework presented here can be developed to study complex microbial systems in other settings, including within the human microbiome.